Nature Communications

Nat Commun. 10: 3393

The MTR4 helicase recruits nuclear adaptors of the human RNA exosome using distinct arch-interacting motifs

Mahesh Lingaraju¹, Dennis Johnsen², Andreas Schlundt³⁴⁵, http://orcid.org/0000-0002-9977-2427Lukas M. Langer¹, Jérôme Basquin¹, http://orcid.org/0000-0002-1594-0527Michael Sattler³⁴, Torben Heick Jensen², http://orcid.org/0000-0001-7848-4621Sebastian Falk

sebastian.falk@univie.ac.at

¹⁶, Elena Conti

conti@biochem.mpg.de

¹

1. 0000 0004 0491 845Xgrid.418615.fDepartment of Structural Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany

2. 0000 0001 1956 2722grid.7048.bDepartment of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Alle 3, 8000 Aarhus C, Denmark

3. 0000000123222966grid.6936.aCenter for Integrated Protein Science Munich (CIPSM) at Department of Chemistry, Technical University of Munich (TUM), 85747 Garching, Germany

4. 0000 0004 0483 2525grid.4567.0Institute of Structural Biology, Helmholtz-Zentrum München, 85764 Neuherberg, Germany

5. 0000 0004 1936 9721grid.7839.5Present Address: Institute for Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ) at Johann Wolfgang Goethe-University, Frankfurt am Main, 60438 Germany

6. 0000 0001 2286 1424grid.10420.37Present Address: Max F. Perutz Laboratories, Department of Structural and Computational Biology, University of Vienna, Campus Vienna Biocenter 5, 1030 Vienna, Austria

© The Author(s) 2019

DOI: 10.1038/s41467-019-11339-x

Published online: 29 July 2019

Abstract

The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins.

Abstract

The human RNA exosome contains a nuclear co-factor MTR4, which unwinds structural RNAs and recruits adaptors for different RNA processing and decay pathways. Here the authors uncover new variations of the arch-interacting motif (AIM) in NVL and ZCCHC8 and characterise their interaction with MTR4.

Introduction

Eukaryotic cells generate a multitude of RNA species that require timely maturation and decay to maintain a healthy transcriptome. A central machinery in nuclear RNA processing, quality control and decay pathways is a conserved 3′–5′ exoribonuclease complex known as the RNA exosome (reviewed in¹,²). Most mechanistic studies to date have analyzed the RNA exosome from S. cerevisiae, the species in which it was originally identified 20 years ago³. The yeast exosome consists of a 10-subunit core complex (Exo₁₀)⁴, the activity of which depends on a single processive exoribonuclease (Rrp44, also known as Dis3)⁵,⁶. The Exo₁₀ core is present in both nuclear and cytoplasmic compartments, but its cofactors and regulators have distinct subcellular localizations (reviewed in¹,²). In the nucleus, Exo₁₀ is bound to the distributive ribonuclease Rrp6 and its associated protein Rrp47 as well as to the Mpp6 protein⁷–⁹. Together, Rrp6-Rrp47 and Mpp6 recruit the RNA helicase Mtr4 to the exosome¹⁰–¹². Orthologues of all these 14 proteins exist in human cells, and engage in similar interactions to form the corresponding human nuclear exosome complex¹²,¹³.

In both yeast and humans, the nuclear helicase Mtr4/MTR4 is central to exosome function²,¹⁴,¹⁵. First, it functions as an enzyme to remodel ribonucleoprotein (RNP) substrates with its 3′–5′ unwinding activity, and to present the unwound RNA substrate to the exosome core¹²,¹⁶. Furthermore, it functions as a binding platform for RNA-binding adaptors, providing the primary interactions to transcripts subjected to exosomal degradation in both RNA processing and decay pathways¹⁷,¹⁸. For example, S. cerevisiae Mtr4 binds Nop53, a ribosome biogenesis factor that recruits the exosome for a late step in rRNA processing, namely the trimming of ITS2 (Internal Transcribed Spacer 2). Two other factors, Trf4 and Air2, bind Mtr4 to form the so-called TRAMP complex¹⁹–²¹, which allows the exosome to target aberrant tRNAs²², rRNAs and small nuclear and nucleolar RNAs (sn/snoRNAs) for decay²³. Higher eukaryotes not only have orthologues of Nop53 and TRAMP, but also have an increased number of nuclear exosome adaptors. In human cells, MTR4 has been reported to interact with the early ribosome biogenesis factors WDR74 and NVL, which take part in the processing of ITS1 (Internal Transcribed Spacer 1)²⁴,²⁵. Human MTR4 also binds to two large Zinc-finger proteins, ZCCHC8 and ZFC3H1. ZCCHC8 interacts with MTR4 and the RNA-binding protein RBM7 to form the trimeric Nuclear EXosome Targeting (NEXT) complex¹⁷,²⁶, which targets enhancer RNAs (eRNAs), promoter upstream transcripts (PROMPTs) and intronic RNAs for exosome-mediated decay²⁷. ZFC3H1 instead directs MTR4 and the nuclear exosome to polyadenylated nuclear RNAs by connecting to the nuclear poly(A) binding protein PABN1²⁸–³⁰. Furthermore, MTR4 binds NRDE-2, a negative regulator that prevents the nuclear exosome  from targeting RNAs that should be exported to the cytoplasm³¹.

How does the nuclear exosome helicase mediate binding to such a diverse and functionally distinct set of proteins? Structural studies have shown that yeast Mtr4 contains an unstructured N-terminal region, a DExH helicase core and an ‘arch’ domain with a globular Kyprides, Ouzounis, Woese (KOW) domain¹⁴,³². All Mtr4 domains are involved in protein-protein interactions: the N-terminal region binds Rrp6-Rrp47¹¹, the helicase core binds Mpp6 as well as Trf4-Air2³³,³⁴ and the KOW domain binds a short sequence known as ‘arch-interacting motif’ that is present in Nop53, Utp18, and Air2³⁵,³⁶. In the case of human MTR4, both the arch domain and the DExH helicase core bind NRDE-2³¹. Structural data have also elucidated how the helicase core of human MTR4 binds a region of ZCCHC8³⁷. This protein is however expected to harbor another MTR4-binding region³⁷, which remains to be identified. Also unclear is how other human MTR4-binding proteins are recognized. With the exception of the expected AIM sequence in the human NOP53 orthologue (also known as GLTSCR2) and in NRDE-2³¹, there is no consensus motif that can be identified with confidence at the sequence level in other MTR4-binding factors. Here, we used a combination of biochemical studies, X-ray crystallography and nuclear magnetic resonance (NMR) experiments to obtain mechanistic insights into how human MTR4 interacts with two metazoan RNA exosome adaptors: the RNA processing factor NVL and the RNA decay factor ZCCHC8.

Results

The unstructured region of human NVL interacts with MTR4

The nuclear VCP-like (NVL) protein, also known as NVL2, is a ribosome biogenesis factor of the AAA-ATPase family that has been reported to interact with human MTR4³⁸. NVL is a multidomain protein characterized by an N-terminal nucleolin-binding domain (residues 10–74)³⁹ and a linker region (residues 76–266) followed by two globular domains, characteristic of AAA-ATPases that are responsible for catalytic activity (Fig. 1a, Supplementary Fig. 1a). Bioinformatic analyses suggested that the linker region is mostly unstructured (residues 76–239) (Supplementary Fig. 1b). In other AAA-ATPases, unstructured regions upstream of the catalytic domains often mediate protein-protein interactions⁴⁰–⁴². To test if the portion upstream of the NVL catalytic domains mediates the interaction with MTR4, we expressed and purified the N-terminal region of human NVL tagged with thioredoxin (Trx) as the prey protein (Trx-NVL1–266) and the structured portion of MTR4 tagged with glutathione-S-transferase (GST) as the bait protein (GST-MTR4-∆N). In these assays, Trx-NVL1–266 specifically co-precipitated with GST-MTR4-∆N (Fig. 1b, lane 11, compare with GST control in lane 9). Interestingly, pull-down assays with the recombinant yeast orthologues showed that the N-terminal unstructured region of Rix7 does not interact with yeast Mtr4 (Supplementary Fig. 1c), indicating that the interaction between human NVL and MTR4 is not conserved in S. cerevisiae.

Fig. 1View larger versionFig. 1. The N-terminal unstructured region of NVL interacts with the MTR4 KOW. a Schematic representation of the domain organization of NVL and MTR4. Domain boundaries are obtained from previous studies³¹,³⁷ and the crystal structure in the current report. The arrows indicate nomenclature for MTR4 constructs used in the text. b Protein co-precipitation assays. GST-tagged MTR4∆N, MTR4∆N∆arch, and MTR4 KOW were incubated with either Trx-NVL1–266 or Trx-NVL167–216 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes 1–8) and 30% of the eluates (lanes 9–16) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. c Microscale thermophoresis experiment with MTR4 KOW and Trx-NVL167–216-(GS)₃-eYFP. In all, 50 nM of Trx-NVL167–216-(GS)₃-eYFP was incubated with increasing concentrations of MTR4 KOW and thermophoresis was measured by tracking the fluorescence of the NVL-YFP fusion protein. The binding isotherm was calculated using MO software (Nanotemper technologies) and the dissociation constant (Kd) is given in the inset. The error bars represent the standard deviations of each data point calculated from three independent thermophoresis measurements

We narrowed down the MTR4-interacting region of human NVL based on bioinformatic analysis. Sequence alignments showed that the N-terminal unstructured region of NVL contains an insertion that is present in the human protein and other chordates but not in the yeast orthologue (Supplementary Fig. 1a). Upon testing whether this insertion is responsible for the interaction with human MTR4, we found that a construct encompassing the conserved portion of the human NVL insertion spanning residues 167–216 (Supplementary Fig. 1a) was indeed able to co-precipitate with GST-MTR4-∆N in pull-down assays (Fig. 1b, lane 14, Trx-NVL167–216). Thus, the MTR4-binding determinants of NVL reside in an unstructured region that is present in the human orthologue and more generally in chordate NVL proteins, but not in lower eukaryotes.

NVL targets the MTR4 KOW domain

To identify where the MTR4-binding determinants for NVL reside, we performed GST pull down assays with MTR4 constructs harboring the DExH core of the helicase (GST-MTR4∆N∆arch) and the KOW domain (GST-MTR4 KOW). Neither Trx-NVL1–266 nor Trx-NVL167–216 co-precipitated with GST-MTR4-ΔNΔarch (Fig. 1b, lanes 12 and 15). In contrast, the KOW domain characteristic of the MTR4 arch (GST-MTR4-KOW) was able to co-precipitate both Trx-NVL1–266 and Trx-NVL167–216 (Fig. 1b, lanes 13 and 16). We quantified the strength of the interaction by biophysical approaches. Using microscale thermophoresis (MST), we measured a dissociation constant of ~1 µM between a YFP-tagged version of NVL167–216 and MTR4-KOW (Fig. 1c). A similar dissociation constant was obtained when testing the interaction of NVL167–216 and MTR4-∆N using isothermal titration calorimetry (ITC) (Supplementary Fig. 1d). The fact that the KOW domain alone binds NVL167–216 as strongly as MTR4-∆N indicated that the NVL167–216 binding region resides within the KOW domain.

We proceeded to characterize the NVL167–216-binding site of MTR4 with structural approaches. Using NMR spectroscopy, we confirmed that the MTR4-KOW domain has a sequential arrangement of secondary structure elements consistent with the fold observed in a recent crystal structure of an MTR4-NRDE-2 complex³¹, namely a five-stranded β-sheet flanked by a long C-terminal helix and containing smaller helical segments within loops (Supplementary Fig. 2). We then carried out titration experiments using a ¹⁵N-labelled MTR4-KOW protein and adding increasing amounts of unlabeled NVL167–216. In line with a dissociation constant of ~1 µM, we observed an intermediate exchange regime for most of the peaks during the titration in HSQC (heteronuclear single quantum coherence) spectra. Chemical shift perturbations (CSP) measured upon NVL2167–216 addition revealed significant effects (Fig. 2a–c) with clusters of strongly shifting peaks around residues 658, 695, 743, and 764. From the NMR analysis of the NVL167–216 - MTR4-KOW interaction, the CSPs were very similar to those we had previously reported for the yeast Nop53-Mtr4 KOW interaction³⁶ (Supplementary Fig. 3a, 3b). Consistently, a reverse-charge substitution of Arg743 (R743E mutant) impaired the binding of both NVL167–216 (pull-down assays in Fig. 2d, lane 8) and of the human Nop53 orthologue (Supplementary Fig. 3c).

Fig. 2View larger versionFig. 2. Analysis of MTR4 KOW-NVL complex by NMR and site directed mutagenesis. a Overlay of ¹H-¹⁵N-HSQCs of either MTR4 KOW alone (black) or in complex with a six-fold excess of NVL2 (red). Selected residues experiencing large chemical shift perturbations (CSPs) are labeled. b Plot of CSPs per residue of the MTR4 KOW sequence. The red line marks the threshold of significant CSPs, which are mapped on the model of MTR4 KOW (PDB 6IEH) in panel c. Gaps indicate either prolines or residues that could not be assigned. c MTR4 KOW domain (PDB 6IEH) with labeled secondary structural elements as assigned by NMR and residues showing significant CSPs (displayed as red/green spheres). d Protein co-precipitations by pull down assays. GST-tagged MTR4∆N (WT or mutants) were incubated with Trx-NVL167–216 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes 1–4) and 30% of the eluates (lanes 5–8) were analyzed on 15% SDS-PAGE gels and visualized by staining with coomassie brilliant blue

Identification of W-AIM: a tryptophan arch-interacting motif of NVL

Given that NVL167–216 binds the KOW domain of MTR4 similarly to Nop53, we expected the presence of a similar AIM motif. A stretch of amino acids within this segment (NVL residues 185–190) appeared to show significant sequence similarity to the Nop53 AIM sequence (Fig. 3a). Surprisingly in this context, the F186A and D189R mutations in the Nop53-like stretch did not affect NVL167–216 binding to MTR4 in a pull-down assay with recombinant proteins (Fig. 3b, compare lanes 5 and 6), indicating that this segment is not a bona-fide AIM. To identify the arch-interacting motif in NVL167–216, we analyzed the sequences from different species in terms of evolutionary conservation. We noticed that vertebrates feature a conserved hydrophobic segment GWFIDKTP (residues 172–179, Fig. 3a; Supplementary Fig. 1a). Mutations in this segment (W173A, I175E) either abolished or impaired NVL167–216 binding to MTR4∆N in pull-down assays in vitro (Fig. 3b, lanes 7 and 8), suggesting that this stretch functions as an arch-interacting motif, which we coin ‘tryptophan-centered arch - interacting motif’ (W-AIM).

Fig. 3View larger versionFig. 3. The vertebrate specific W-AIM in NVL is crucial for binding to MTR4 KOW. a Sequence alignment of vertebrate specific insertion regions of representative NVL sequences, Homo sapiens (Hs), Gallus gallus (Gg), Xenopus tropicalis (Xt), highlighting the W-AIM (GWFIDKTP) (red box), and the Nop53-like region (LFXϕD) (purple). The sequences were obtained from the UniProt database and aligned using the T-coffee server⁵⁹. b Protein co-precipitations by pull down assays. GST-tagged MTR4∆N was incubated with either wild type Trx-NVL167–216 or its indicated mutant before co-precipitation with glutathione sepharose beads. A total of 3% of the input (top) and 30% of the eluates (bottom) were analyzed on 15% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. c Overall structure of MTR4∆N₂70–1042 - NVL167–216 complex, with the DExH core of MTR4 colored in gray and arch colored in light blue. NVL is colored in orange. d Zoom-in view of the interactions between MTR4 KOW (light blue) domain and NVL (orange). Domains are colored as in Fig. 3a and viewed are 90° rotation along the horizontal and vertical axes with respect to the view in Fig. 3a. Residues discussed in the text are highlighted and labeled. e Cellular co-IP assay. FLAG-tagged wild type NVL construct, or its indicated mutant variant, were transiently expressed in cells stably expressing MTR4-LAP. After precipitation of MTR4-LAP, a total of 0.5% of the input (left) and 8.0% of the eluates (right) were analyzed on 4–12% SDS-PAGE gel followed by western blotting analysis. The primary antibody used is indicated below the panel

We co-crystallized the NVL W-AIM peptide in complex with a construct of human MTR4 encompassing both the DExH core and the arch domain (residues 70–1042) (Table 1). The overall structure of the helicase is similar to that of the human NRDE-2-MTR4 complex³¹, but with a notably different conformation of the arch domain. In the NVL-bound crystal structure, the arch domain of MTR4 is an open conformation, with the KOW domain clearly separated from the helicase core (Fig. 3c and Supplementary Fig. 4a). The NVL W-AIM polypeptide chain binds in an extended conformation, similar to that reported for NRDE-2³¹, Nop53³⁶ and Air2³⁴ (Supplementary Fig. 4b, 4c). The hydrophobic side chains of Trp173NVL, Phe174NVL, and Ile175NVL contact Val766MTR4, Tyr745MTR4, and Val742MTR4 respectively, while Asp176NVL interacts with Arg743MTR4 (Fig. 3d). The NVL polypeptide chain then forms a pronounced bend at Pro179NVL (which contacts Phe653MTR4) and continues to form a crystal contact, docking at the Trf4-binding site of a symmetry related helicase core (Supplementary Fig. 5a). Consistent with the structural data, mutation of Asp176NVL (D176A mutant) impairs the binding of NVL167–216 to MTR4∆N in a GST pull down experiment (Supplementary Fig. 5b).

See full tableTable 1. Data collection and refinement statistics

Finally, we tested the effect of the MTR4-interacting residues of NVL in human cells. To this end, we carried out co-immunoprecipitation (co-IP) assays in HeLA cells stably expressing MTR4 with an eGFP ‘localization and affinity purification’ (LAP) tag at the N-terminus and transiently expressing FLAG-tagged full-length NVL constructs (wild type, or with the W173A/I175A mutation or with deletion of the entire hydrophobic segment). Western blotting analysis confirmed that wild type NVL bound MTR4 and that disruption of the 172–180 segment, either by mutation or deletion, impaired the NVL-MTR4 interaction (Fig. 3e, compare lanes 8, 9, 10). Taken together, we conclude that NVL interacts with the KOW domain of MTR4 using a short linear W-AIM sequence. The NVL W-AIM is more hydrophobic than the Nop53 AIM³⁵, rationalizing why it binds MTR4 with an order of magnitude higher affinity than the yeast Nop53-Mtr4³⁶ and human Nop53-MTR4 interactions (Supplementary Fig. 3d).

The unstructured region of ZCCHC8 interacts with the MTR4 KOW domain

Identification of the W-AIM sequence in NVL motivated us to examine whether other known MTR4 interactors also contain a similar tryptophan-centered motif. One such interactor is ZCCHC8, the scaffolding subunit of the NEXT complex²⁷,³⁷,⁴³. This modular protein contains a predicted N-terminal coiled–coil domain (residues 40–80), a Zinc-knuckle domain (residues 222–246), a proline-rich domain (residues 287–334) that interacts with RBM7⁴³ and a C-terminal domain (CTD) (residues 659–707) (Fig. 4a) that interacts with the DExH core of MTR4 and activates it³⁷. The N-terminal portion of ZCCHC8 is expected to contain an additional MTR4-binding site³⁷. Within the N-terminal portion, we focused our attention on the linker between the coiled-coil and the Zinc-knuckle domains, as it appeared to contain an NVL-like tryptophan-containing sequence (Supplementary Fig. 6a). We expressed a large portion of this linker (residues 91–211) tagged to maltose binding protein (MBP-ZCCHC891–211) and purified the resulting construct for pull-down assays with the versions of GST-tagged MTR4 described above (Fig. 1a). MBP-ZCCHC891–211 co-precipitated with GST-MTR4-∆N but not with GST-MTR4-∆N∆arch (Fig. 4b, lanes 6 and 7). Similar to the results we had obtained for NVL167–216, the pull-down assays showed that MBP-ZCCHC891–211 interacts with GST-MTR4-KOW (Fig. 4b, lane 8). Next, we determined the affinity of the MTR4-KOW-ZCCHC891–211 interaction using microscale thermophoresis (MST) with a fluorescent-tagged version of ZCCHC891–211 (that we had engineered by fusing a YFP to the C-terminus). In this quantitative assay, we measured a dissociation constant (KD) of ~0.3 µM (Fig. 4c), indicating a higher affinity than that of the NVL167–216 fragment. To corroborate these results, we pre-formed a GST-MTR4-∆N - Trx-NVL167–216 complex and incubated it with increasing amounts of MBP-ZCCHC891–211 before subjecting the mixtures to GST pull-down assays. The competition assay showed that ZCCHC891–211 could displace NVL167–216 from the pre-formed complex (Fig. 4d, lanes 5–8), suggesting that they interact with the exosome helicase in a mutually exclusive manner.

Fig. 4View larger versionFig. 4. The N-terminus of ZCCHC8 interacts with the MTR4 KOW domain. a Schematic representation of the domain organization of ZCCHC8. Domain boundaries are obtained from previous studies³⁷,⁴³ and computational sequence analysis. b Protein co-precipitations by pull down assays. GST-tagged MTR4∆N, MTR4∆N∆arch, and MTR4 KOW were incubated with MBP-ZCCHC8 91–211 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes 1–4) and 30% of the eluates (lanes 5–8) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. c Microscale thermophoresis experiment with MTR4 KOW and MBP-ZCCHC8 91–211-(GS)₃-eYFP. In all, 1 μM MBP-ZCCHC8 91–211-(GS)₃-eYFP was incubated with increasing concentrations of MTR4 KOW and thermophoresis was measured by tracking the fluorescence of the ZCCHC8-YFP fusion protein. The binding curve was calculated using MO software (Nanotemper technologies) and the dissociation constant (Kd) is given in the inset. The error bars represent the standard deviations of each data point calculated from three independent thermophoresis measurements. d Competition experiment using GST pull down assay technology. A preformed GST-MTR4∆N:Trx-NVL167–216 complex was incubated with increasing concentrations of MBP-ZCCHC8(91–211) before precipitation with glutathione sepharose beads. A total of 3% of the input (lanes, 1–4) and 30% of the eluates (5–8) were analyzed on 15% SDS-PAGE gels and visualized by staining with coomassie brilliant blue

Identification of C-AIM: a cysteine arch-interacting motif of ZCCHC8

We proceeded to identify the MTR4-binding site within the N-terminal region of ZCCHC8. We first interrogated the NVL-like tryptophan-containing patch (GWEIPK, residues 197–202, Fig. 5a). Surprisingly, mutation of Trp198 and Lys202 (W198A/K202E mutant) did not alter the interaction of ZCCHC891–211 with MTR4-∆N in GST pull down assays (Fig. 5b, lane 7), indicating that this patch of ZCCHC8 is not a bona-fide W-AIM sequence.

Fig. 5View larger versionFig. 5. Analysis of the ZCCHC8-MTR4 KOW complex by site directed mutagenesis. a Sequence alignment of the region between the predicted coiled coil domain and the zinc finger of ZCCHC8 from representative metazoan species, Homo sapiens (Hs), Gallus gallus (Gg), Xenopus laevis (Xl), highlighting NVL-like region (red), Nop53-like AIM (C-AIM) and ZCCHC8 specific I-AIM (purple boxes). The sequences were obtained from the UniProt database and aligned using the T-coffee server⁵⁹. b Protein co-precipitations by pull down assays testing ZCCHC8 91–211 mutants for MTR4∆N binding ability. GST-tagged MTR4∆N was incubated with either ZCCHC891–211 WT or mutants before co-precipitation with glutathione sepharose beads. A total of 3% of the input (top) and 30% of the eluates (bottom) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. c Protein co-precipitations by pull down assays testing MTR4∆N mutants for their ZCCHC8 91–211 binding ability. GST-tagged MTR4∆N (WT or mutant variants) were incubated with MBP-ZCCHC8 91–211 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes, 1–4) and 30% of the eluates (5–8) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. d Cellular co-IP assay. FLAG-tagged ZCCHC8 constructs (WT/IF mutant/CTD deletion) were transiently expressed in cells stably expressing MTR4-LAP. After precipitation of MTR4 taking advantage of the LAP tag, a total of 0.5% of the input (left) and 8.0% of the eluates (right) were analyzed on 4–12% SDS-PAGE gel followed by western blotting analysis. The primary antibody used is indicated below the panel

To identify the arch-interacting motif in ZCCHC891–211, we took a similar bioinformatics approach as described above for NVL. When analyzing the evolutionary conservation of ZCCHC891–211, we identified a conserved patch centered at the FCLDKLG segment (residues 178–184, Fig. 5a). Mutation of Phe178 and Asp181 (F178A/D181R mutant) in this segment impaired the interaction with MTR4-∆N (Fig. 5b, lane 6). This cysteine-centered arch-interacting motif (C-AIM) is loosely related to the Nop53 AIM and the NVL W-AIM segments, and is thus expected to bind to the same site of the KOW domain. Consistently, the Arg743 reverse-charge substitution (R743E) in MTR4 also impaired the binding of ZCCHC891–211 (Fig. 5c, lane 8).

ZCCHC8 also contains a non-canonical arch-interacting motif: I-AIM

While attempting to narrow down the KOW binding region of ZCCHC8 further, we observed that N-terminal truncation of ZCCHC891–211 resulted in a near loss of MTR4 binding (Supplementary Fig. 6b). Based on these results, we reasoned that ZCCHC891–211 might harbor an additional MTR4-binding motif that would be predicted to bind to an adjacent surface on the KOW domain (and thus show no resemblance to the previously identified arch-interacting motifs). Using bioinformatics approaches, we identified another conserved segment upstream of C-AIM (IEEF, residues 112–115). Indeed, mutation of Ile112 and Phe115 (I112R/F115R mutant) severely weakened the MTR4-∆N - ZCCHC891–211 interaction in GST pull-down assays (Fig. 5b, lane 8). Thus, this segment (which we refer to as an isoleucine-centered arch-interacting motif, or I-AIM) also contributes to MTR4 binding. Next, we mapped the possible MTR4-binding site of I-AIM. The β-barrel of the MTR4 KOW domain is structurally related to that of Tudor domains (Supplementary Fig. 7a), small globular folds that generally present a hydrophobic pocket for binding methylated arginines and lysines⁴⁴. Although there is little overall sequence similarity, MTR4-KOW features hydrophobic residues at the equivalent position of the substrate–binding residues of Tudor domains (Supplementary Fig. 7b)⁴⁴,⁴⁵. In particular, MTR4 Phe677 is evolutionarily conserved (Supplementary Fig. 7b) and is located on a surface adjacent to the Arg743 site where the Nop53 like AIM, W-AIM and C-AIM sequences are recognized (Supplementary Fig. 7c). In line with this site being used for the additional I-AIM sequence of ZCCHC891–211, mutation of Phe677 (F677E) disrupted binding of ZCCHC891–211 (Fig. 5c, lane 7) and did not affect the binding of NVL167–216 (Fig. 2d, lane 7) or human Nop53 (Supplementary Fig. 3c, lane 7).

We tested the importance of the I-AIM segment in human cells. HeLa LAP-MTR4 cells were transfected with FLAG-tagged full-length ZCCHC8 constructs (wild type, the I-AIM I112R/F115R double mutant or a deletion construct lacking the ZCCHC8 CTD). Western blotting analysis of the resulting co-IPs revealed that disrupting the I-motif by mutation is sufficient to impair the ZCCHC8-MTR4 interaction, whereas deletion of the CTD did not significantly affect complex formation (Fig. 5d, lanes 8, 9, 10). These mutations also did not affect the ZCCHC8-RBM7 interaction (Supplementary Fig. 6c). The CTD of ZCCHC8 has been shown to bind the DExH core of MTR4 and to enhance helicase activity³⁷. In contrast, we found that neither the ZCCHC8 nor the NVL AIM motifs have a significant effect on the catalytic properties of MTR4, as judged by assaying both ATP hydrolysis and RNA helicase activities (Supplementary Fig. 8). These results suggest a division of labor of the N-terminal and C-terminal ZCCHC8 regions: while the C-terminal domain regulates the activity of the helicase³⁷, the N-terminal region plays has a central scaffolding role in incorporating MTR4 into the NEXT complex.

Discussion

In this study, we show that the human nuclear exosome adaptors NVL and ZCCHC8 bind the MTR4 KOW domain on a surface that is also employed by Nop53³⁶ and NRDE-2³¹ using distinct arch-interacting motifs³⁵. The AIMs of Nop53/Utp18/Air2 (LFxϕD(x)1–2G/P), NVL (GWFIDKTP), ZCCHC8 (NFCLDKLG), and NRDE-2 (SFRTDKKP) can best be considered as subfamilies of canonical AIMs. With insight, sequences in both NVL and ZCCHC8 that at first glance appeared to resemble known AIMs instead contain individual amino acids that are likely to prevent MTR4 binding. The consensus sequence of canonical bona-fide AIMs can thus be re-defined as xωxxD(x)1/2G/P, with a C-terminal glycine or proline residue that allows the polypeptide chain to bend as it extends away from its binding site. The aromatic (ω) and polar/non-polar amino acids might vary, but tend to be conserved within individual subfamilies of canonical AIMs. Despite the variability, all canonical AIM sequences are recognized, in a mutually exclusive manner, at a surface pocket of the MTR4 KOW domain that is defined by the presence of Arg743 (yeast Mtr4 Arg774). Mutation of this surface pocket may thus be a useful tool to probe new MTR4-interacting proteins that contain arch-interacting motifs, as these motifs are difficult to identify due to the degeneracy of their consensus sequence. Furthermore, we found that ZCCHC8 harbors an additional arch-interacting motif that does not conform to the canonical AIM consensus sequences. These findings show how different exosome adaptor proteins have evolved similar mechanisms to recognize MTR4 in a specific and mutually exclusive manner, but can also modulate the affinity and thus selectivity with which they are ultimately recruited to the nuclear exosome.

Methods

Protein expression and purification

Human MTR4 constructs (full-length, MTR4-∆N (75–1042), MTR4-KOW (645–787) and MTR4-∆N∆arch, where residues 598–842 were substituted by 2xGS linker) were expressed as 6xHis-GST-tagged fusion (cleavable with 3 C protease) proteins in BL21 star (DE3) E. coli cells, grown either in terrific broth (for biochemical studies) or minimal medium supplemented with ¹⁵N labelled ammonium chloride and/or ¹³C labeled Glucose (for NMR studies). The proteins were purified using a Ni-nitrilotriacetate (NTA) affinity column and a heparin column (GE healthcare) for ion exchange chromatography. When appropriate, the His-GST tag was cleaved at this point by incubation with 3 C protease, followed by removal of the tag with an additional Ni-NTA affinity step. Finally, the protein was subjected to size exclusion chromatography on a Superdex 200 column (GE healthcare) in 50 mM Hepes/NaOH pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, 2 mM DTT.

The human MTR4 construct used for crystallization (71–1042) was cloned as GST fusion construct with a TEV cleavage site immediately preceding the MTR4 sequence which when cleaved would leave a glycine (the natural 70th residue in MTR4) yielding MTR4 protein with residues 70–1042 (referred to as MTR4∆N₂ in the text). The construct was purified as described above.

The primers used for cloning all the constructs described are listed in Supplementary table 1.

All NVL and ZCCHC8 constructs and truncation mutants described in the text were expressed as 6xHis-Trx and 6xHis-MBP tagged fusion (cleavable with 3 C protease) proteins, respectively in BL21 star (DE3) E. coli cells. The proteins were purified using a Ni-NTA affinity column. When appropriate, the tags were cleaved upon incubation with 3 C protease at this step, followed by an additional Ni-NTA affinity step for the removal of the tag. In case of ZCCHC8, the tag cleavage was performed only in the presence of MTR4 to prevent the protein from precipitating. In the final purification step, the proteins were subjected to size-exclusion chromatography on a Superdex 75 in 50 mM Hepes/NaOH pH 7.5, 150 mM NaCl, 2 mM DTT.

Biophysical assays

The microscale thermophoresis measurements were performed on a NanoTemper Monolith NT.115 machine. Before the measurements, all samples were dialyzed against a buffer containing in 50 mM Hepes/NaOH pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, 0.5 mM TCEP. For NVL, 50 nM of Trx-NVL167–217-(GS)₃-eYFP was incubated with increasing concentrations of unlabeled MTR4 KOW and thermophoresis was measured with an MST power of 20% and an LED power of 20%. For ZCCHC8, 1 μM of MBP-ZCCHC891–211 -(GS)₃-eYFP was incubated with increasing concentrations of unlabeled MTR4-KOW and thermophoresis was measured with an MST power of 70% and an LED power of 15%. In both cases, the MTR4 KOW concentration series was produced by serial dilution (1:1). Titrations were performed in triplicates and the data were analyzed using the Thermophoresis with T-Jump strategy option with the MO software (NanoTemper Technologies).

Isothermal calorimetry experiments were carried out using the ITC200 Isothermal titration calorimeter from Microcal. Before the measurement, all samples were dialysed against a buffer containing 50 mM Hepes/NaOH pH 7.5, 150 mM NaCl, and 0.5 mM TCEP. For NVL: MTR4-∆N (the reactant) samples were concentrated to 40 µM and NVL (the injectant) to 500 µM. For human NOP53: MTR4-KOW (the reactant) samples were concentrated to 100 µM and NOP53 (the injectant) to 1 mM. Titrations were carried out at 25 °C with 2 µL of the injectant per injection added to 200 µL of reactant cell solution. All data were processed and curves fitted using Origin5.0.

Biochemical assays

For pull-down assays, appropriate protein mixtures were incubated in 50 mM Hepes (pH 7.5), 150 mM NaCl, 5% (v/v) glycerol, 0.05% (v/v) NP40, 1 mM DTT for 30 min at 4 °C. For ZCCHC8, 2 μM GST-MTR4∆N was incubated with 8 μM MBP-ZCCHC8 constructs. For NVL, 10 μM GST-MTR4∆N was incubated with 20 μM Trx-NVL constructs. For NOP53, 30 μM GST-MTR4∆N was incubated with 60 μM Trx-NOP5384–123 in a total volume of 30 μl. The protein mixtures were then incubated with Glutathione sepharose beads (GE healthcare) for 2 h. Post incubation, the beads were washed three times with 0.1 ml incubation buffer and the retained material was eluted with incubation buffer supplemented with 30 mM reduced glutathione. Input material (1–3%) and eluates (~30%) were analyzed by SDS-PAGE and Coomassie staining.

For ATPase assays, 150 pmol of MTR4∆N or MTR4∆N-containing complexes were incubated 40 nmol MESG (2-amino-6-mercapto-7-methylpurine ribonucleoside) and 0.5 U purine nucleoside phosphorylase (Enzchek Phosphate Assay kit, Invitrogen) in a buffer containing 50 mM MOPS pH 6.5, 50 mM Nacl, 2.5 mM MgCl₂, 5 mM β-mercaptoethanol and 5% (v/v) glycerol. For reactions containing RNA, the mixture was incubated with 2 μg poly-U RNA (Sigma). The reaction was initiated by addition of ATP to a final concentration of 1 mM. The generation of 2-amino-6-mercapto-7-methylpurine from MESG was monitored by measuring absorbance increase at 360 nm on a plate reader (Infinite M1000 Pro, Tecan) for 12 min at 60 s intervals. The data were normalized by subtracting the y-intercept from the raw data. The experiment was performed in triplicate. The mean (n = 3) and standard deviation (error bars) were plotted using Graphpad prism 8.

Helicase assays were performed essentially as described by Puno et al.³⁷. A duplex RNA was formed by mixing a 5’ Fluorescein amidite (FAM) labeled RNA (FAM-AGCACCGUAAAGACGC) with 1.5 molar excess of complementary RNA with a 25 A overhang (GCGUCUUUACGGUGCUAAAAAAAAAAAAAAAAAAAAAAAAA) in a buffer containing 20 mM Tris-HCl pH 7.5 and 50 mM NaCl. To anneal the RNA duplex, the mixture was heated to 95 °C and allowed to cool down slowly to ambient temperature by turning off the heat block. 0.3 pmol of duplex RNA was incubated with 3.75, 7.5, 30 pmol of MTR4∆N or MTR4∆N- containing complexes in a buffer containing 50 mM MOPS pH 6.5, 50 mM Nacl, 0.5 mM MgCl₂, 5 mM β-mercaptoethanol and 5% (v/v) glycerol for 5 min at 30 °C. The reaction was then initiated by the addition of ATP, MgCl₂ and a trap DNA oligo complementary to the FAM labeled RNA (GCGTCTTTACGGTGCT) to a final concentration of 2 mM, 2 mM and 400 nM respectively. The reactions were quenched after 40 min by placing the tubes on ice and adding quenching buffer to a final concentration of 0.5% (w/v) SDS, 10 mM EDTA, 10% (v/v) glycerol and 0.005% (w/v) xylene cyanol. The sample were analyzed by electrophoresis on a 15% acrylamide–Tris base, boric acid, EDTA (TBE) gel. The fluorescence was imaged using a Typhoon FLA 7000. The oligonucleotides used in the assay were obtained from Ella Biotech GmBH.

NMR spectroscopy

NMR measurements of MTR4-KOW were performed in phosphate buffered saline (10 mM PO₄³⁻, 137 mM NaCl, 2.7 mM KCl) mixed with 10% (v/v) D₂O. Backbone chemical shift assignments of the KOW region were obtained from two ¹³C, ¹⁵N-labelled samples with protein concentration of 700 μM and 500 μM, respectively. HNCA, HNCACB, HNcoCA, HNCO, HNcaCO, and 3D ¹⁵N-edited NOESY spectra⁴⁶ were acquired at 298 K on Bruker Avance III spectrometers at field strengths corresponding to 600 and 800 proton Larmor frequency, equipped with TCI cryogenic probe heads. The ¹⁵N steady-state heteronuclear {¹H}-¹⁵N NOE experiment was performed at 170 μM and a field strength of 500 MHz as described previously⁴⁷. Protein binding was measured from HSQC experiments containing water-flip-back/WATERGATE⁴⁸,⁴⁹ sequences. Titrations with the NVL peptide were carried out at 298 K with a KOW concentration of 53 μM and in presence of 0.25, 0.5, 1, 2, and 6 stoichiometric molar equivalents of NVL. Spectra were recorded and processed with Topspin3.5 and analyzed with CCPNMR Analysis 2.4⁵⁰ and Sparky (http://www.cgl.ucsf.edu/home/sparky). The chemical shift perturbations were calculated as CSP (ppm) = [6(ΔH)² + (ΔN)²]0.5.

Crystallization and structure determination

MTR4∆N₂ (70–1042) was mixed with 2 molar excess of NVL (167–216) in a buffer containing 20 mM Tris pH 7.5, 150 mM Nacl, 1 mM Mgcl₂, 2 mM ADP and 1 mM TCEP. The crystallization trials were performed using a vapour diffusion setup. Initial crystals were obtained in the A4 (2 M ammonium sulfate, 0.1 M Tris pH 8.5) condition of SG1TM screen (Molecular dimensions). The best diffracting crystals were obtained at a concentration of 10 mg/ml at 277 K in 0.1 M Tris pH 8, 1.8 M ammonium sulfate. The crystals were cryo-protected with reservoir solution supplemented with 30% glycerol prior flash-freezing in liquid nitrogen. Data were collected at 100 K at PXIII beamline of the Swiss light Source (Villigen, Switzerland). Data processing and scaling was performed using Xia2/DIALS⁵¹,⁵² within CCP4i2 software suite⁵³. The crystals belong to the hexagonal spacegroup 169 (P6₁) containing one molecule (MTR4∆N₂ – NVL167–216) in the asymmetric unit and diffract to 3.07 Å resolution. The structure was solved by molecular replacement with Phaser⁵⁴ within Phenix using the co-ordinates of the DExH core (98-593 and 847–1042) and KOW domain (645–787) of MTR4 (PDB 6IEH)³¹ as search models. The model was manually completed in COOT⁵⁵ and refined using phenix.refine⁵⁶. The optimal TLS groups for TLS refinement were determined using TLSMD server⁵⁷. 96.1% of the protein backbone dihedral angles in the final model are in Ramachandran favored region. The figures of crystallographic models were prepared using pyMOL (Schrödinger, LLC).

Cell culture and co-immunoprecipitation assays

Human HeLa Kyoto LAP, MTR4-LAP, RBM7-LAP cell lines were generated as outlined by Poser et al.⁵⁸ and used for example by Lubas et al.²⁷ (RBM7-LAP) and Meola et al.³⁰ (MTR4-LAP). Briefly, HeLa Kyoto LAP MTR4-LAP, RBM7-LAP cells were cultured in DMEM + 10% FBS. Transfection was carried out using 10 µg of plasmid containing FLAG-tagged NVL or ZCCHC8 constructs, with Lipofectamine 2000 (Thermo Fisher) following the manufacturer’s instructions. Forty-eight hours after transfection the cells were collected and resuspended in extraction buffer (150 mM NaCl, 20 mM HEPES pH 7.4, 0.5% (v/v) Triton X-100) containing Protease Inhibitors (Roche). The lysates were sonicated twice for 5 s at 20 W, and cell debris was removed by centrifugation at 10,000 g for 10 min. Lysates were incubated 1 h with Dynabeads M-270 Epoxy (Invitrogen) coupled to a polyclonal llama anti-GFP antibody. Beads were washed three times in extraction buffer, then incubated 20 min at 25 °C with 100 units of Benzonase (Sigma) and 2 mM MgCl₂. Beads were then washed twice in extraction buffer and proteins were eluted using NuPage LDS Sample Buffer (Invitrogen) at 70 °C for 10 min, and NuPage Sample Reducing Agent was added. The input material (0.5%) and the eluate (8.0%) were analyzed by SDS-PAGE on a NuPage Novex 4–12% Bis-Tris gel (Invitrogen). Western blotting analysis was performed following standard protocols. Following primary antibodies were used: anti-FLAG M2 (Dilution 1:50000; Sigma, F1804), anti-MTR4 (Dilution 1:4000; Abcam, ab70551), anti-GFP (Dilution 1:1000; Santa Cruz Biotechnology, SC-9996), anti-β-actin (Dilution 1:100000; Sigma, A2228) and anti-RBM7 (Dilution 1:1000; human protein atlas, HPA013993). Anti-mouse and anti-rabbit secondary antibodies coupled to Horseradish Peroxidase (Dako) were used.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Supplementary Material

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Footnotes

Peer review information: Nature Communications thanks David Tollervey, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Dennis Johnsen, Andreas Schlundt.

Supplementary information

Supplementary Information accompanies this paper at 10.1038/s41467-019-11339-x.

Acknowledgements

We would like to thank the Crystallization Facility of MPI Biochemistry and also Vincent D. Maciej for his contributions at the early stages of this project. This study was supported by the Max Planck Gesellschaft, the European Commission (ERC Advanced Investigator Grant 294371) and the Deutsche Forschungsgemeinschaft (SFB646, SFB1035, GRK1721, FOR1680 to E.C., and SFB1035 and GRK1721 to M.S. This work was supported by the Cluster of Excellence EXC114 (to E.C. and M.S.). T.H.J. was supported by the ERC (grant 339953), the Lundbeck- and the Novo Nordisk-Foundations.

Author contributions

S.F and E.C. initiated the project; M.L. performed the in vitro experiments; D.J. performed the co-IP experiments under the supervision of T.H.J.; M.L. and J.B. performed the crystallography experiments, M.L. and S.F. built, refined and analyzed the structure; A.S. and M.S. collected and analyzed NMR data; L.L. performed preliminary in vitro experiments with NVL under the supervision of S.F.; M.L., S.F., and E.C. prepared the paper.

Data availability

A reporting summary for this Article is available as a Supplementary Information file. NMR backbone chemical shifts of the human MTR4 KOW domain were deposited at the BMRB under accession number 27831. The coordinates and the structure factors have been deposited in the Protein Data Bank with accession code PDB ID 6RO1. The source data are provided in Supplementary Fig. 10. All data is available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

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References

Chlebowski A, Lubas M, Jensen TH, Dziembowski A, authors. RNA decay machines: the exosome. Biochim Biophys. Acta. 2013;1829:552–560. [CrossRef] [PubMed]

Zinder JC, Lima CD, authors. Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors. Genes Dev. 2017;31:88–100. [CrossRef] [PubMed]

Mitchell P, Petfalski E, Shevchenko A, Mann M, Tollervey D, authors. The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′–>5′ exoribonucleases. Cell. 1997;91:457–466. [CrossRef] [PubMed]

Makino DL, Halbach F, Conti E, authors. The RNA exosome and proteasome: common principles of degradation control. Nat. Rev. Mol. Cell Biol. 2013;14:654–660. [CrossRef] [PubMed]

Dziembowski A, Lorentzen E, Conti E, Seraphin B, authors. A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat. Struct. Mol. Biol. 2007;14:15–22. [CrossRef] [PubMed]

Liu Q, Greimann JC, Lima CD, authors. Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell. 2006;127:1223–1237. [CrossRef] [PubMed]

Butler JS, Mitchell P, authors. Rrp6, Rrp47 and cofactors of the nuclear exosome. Adv. Exp. Med Biol. 2010;702:91–104. [CrossRef] [PubMed]

Milligan L, et al., authors. A yeast exosome cofactor, Mpp6, functions in RNA surveillance and in the degradation of noncoding RNA transcripts. Mol. Cell Biol. 2008;28:5446–5457. [CrossRef] [PubMed]

Schilders G, Raijmakers R, Raats JM, Pruijn GJ, authors. MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation. Nucleic Acids Res. 2005;33:6795–6804. [CrossRef] [PubMed]

Falk S, Bonneau F, Ebert J, Kogel A, Conti E, authors. Mpp6 incorporation in the nuclear exosome contributes to RNA channeling through the Mtr4 helicase. Cell Rep. 2017;20:2279–2286. [CrossRef] [PubMed]

Schuch B, et al., authors. The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase. EMBO J. 2014;33:2829–2846. [CrossRef] [PubMed]

Weick EM, et al., authors. Helicase-dependent RNA decay illuminated by a Cryo-EM structure of a human nuclear RNA exosome-MTR4 complex. Cell. 2018;173:1663–1677 e21. [CrossRef] [PubMed]

Gerlach, P. et al. Distinct and evolutionary conserved structural features of the human nuclear exosome complex. Elife7, e38686 (2018).

Jackson RN, et al., authors. The crystal structure of Mtr4 reveals a novel arch domain required for rRNA processing. EMBO J. 2010;29:2205–2216. [CrossRef] [PubMed]

Kilchert C, Wittmann S, Vasiljeva L, authors. The regulation and functions of the nuclear RNA exosome complex. Nat. Rev. Mol. Cell Biol. 2016;17:227–239. [CrossRef] [PubMed]

Johnson SJ, Jackson RN, authors. Ski2-like RNA helicase structures: common themes and complex assemblies. RNA Biol. 2013;10:33–43. [CrossRef] [PubMed]

Lubas M, et al., authors. Interaction profiling identifies the human nuclear exosome targeting complex. Mol. Cell. 2011;43:624–637. [CrossRef] [PubMed]

Ogami, K., Chen, Y. & Manley, J. L. RNA surveillance by the nuclear RNA exosome: mechanisms and significance. Noncoding RNA4, 8 (2018).

Vanacova S, et al., authors. A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol. 2005;3:e189 [CrossRef] [PubMed]

LaCava J, et al., authors. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell. 2005;121:713–724. [CrossRef] [PubMed]

Wyers F, et al., authors. Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell. 2005;121:725–737. [CrossRef] [PubMed]

Kadaba S, Wang X, Anderson JT, authors. Nuclear RNA surveillance in Saccharomyces cerevisiae: Trf4p-dependent polyadenylation of nascent hypomethylated tRNA and an aberrant form of 5S rRNA. RNA. 2006;12:508–521. [CrossRef] [PubMed]

Carneiro T, et al., authors. Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of polyadenylated RNAs in a discrete domain within the nucleolus. Mol. Cell Biol. 2007;27:4157–4165. [CrossRef] [PubMed]

Hiraishi N, Ishida Y, Nagahama M, authors. AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74. Biochem Biophys. Res Commun. 2015;467:534–540. [CrossRef] [PubMed]

Hiraishi N, Ishida YI, Sudo H, Nagahama M, authors. WDR74 participates in an early cleavage of the pre-rRNA processing pathway in cooperation with the nucleolar AAA-ATPase NVL2. Biochem Biophys. Res Commun. 2018;495:116–123. [CrossRef] [PubMed]

Gustafson MP, Welcker M, Hwang HC, Clurman BE, authors. Zcchc8 is a glycogen synthase kinase-3 substrate that interacts with RNA-binding proteins. Biochem Biophys. Res Commun. 2005;338:1359–1367. [CrossRef] [PubMed]

Lubas M, et al., authors. The human nuclear exosome targeting complex is loaded onto newly synthesized RNA to direct early ribonucleolysis. Cell Rep. 2015;10:178–192. [CrossRef] [PubMed]

Ogami K, et al., authors. An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression. Genes Dev. 2017;31:1257–1271. [CrossRef] [PubMed]

Silla T, Karadoulama E, Makosa D, Lubas M, Jensen TH, authors. The RNA exosome adaptor ZFC3H1 functionally competes with nuclear export activity to retain target transcripts. Cell Rep. 2018;23:2199–2210. [CrossRef] [PubMed]

Meola N, et al., authors. Identification of a nuclear exosome decay pathway for processed transcripts. Mol. Cell. 2016;64:520–533. [CrossRef] [PubMed]

Wang Jianshu, Chen Jiyun, Wu Guifen, Zhang Hongling, Du Xian, Chen Suli, Zhang Li, Wang Ke, Fan Jing, Gao Shuaixin, Wu Xudong, Zhang Shouxiang, Kuai Bin, Zhao Peng, Chi Binkai, Wang Lantian, Li Guohui, Wong Catherine C.L.; Zhou Yu, Li Jinsong, Yun Caihong, Cheng Hong, authors. NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction. Genes & Development. 2019;33(9-10):536–549. [CrossRef] [PubMed]

Weir JR, Bonneau F, Hentschel J, Conti E, authors. Structural analysis reveals the characteristic features of Mtr4, a DExH helicase involved in nuclear RNA processing and surveillance. Proc. Natl Acad. Sci. USA. 2010;107:12139–12144. [CrossRef] [PubMed]

Schuller JM, Falk S, Fromm L, Hurt E, Conti E, authors. Structure of the nuclear exosome captured on a maturing preribosome. Science. 2018;360:219–222. [CrossRef] [PubMed]

Falk S, et al., authors. The molecular architecture of the TRAMP complex reveals the organization and interplay of its two catalytic activities. Mol. Cell. 2014;55:856–867. [CrossRef] [PubMed]

Thoms M, et al., authors. The exosome is recruited to RNA substrates through specific adaptor proteins. Cell. 2015;162:1029–1038. [CrossRef] [PubMed]

Falk S, et al., authors. Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the preribosomal protein Nop53. RNA. 2017;23:1780–1787. [CrossRef] [PubMed]

Puno MR, Lima CD, authors. Structural basis for MTR4-ZCCHC8 interactions that stimulate the MTR4 helicase in the nuclear exosome-targeting complex. Proc. Natl Acad. Sci. USA. 2018;115:E5506–E5515. [CrossRef] [PubMed]

Nagahama M, et al., authors. The AAA-ATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1. Biochem. Biophys. Res Commun. 2006;346:1075–1082. [CrossRef] [PubMed]

Fujiwara Y, et al., authors. Structure and function of the N-terminal nucleolin binding domain of nuclear valosin-containing protein-like 2 (NVL2) harboring a nucleolar localization signal. J. Biol. Chem. 2011;286:21732–21741. [CrossRef] [PubMed]

Meyer H, Bug M, Bremer S, authors. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 2012;14:117–123. [CrossRef] [PubMed]

Hanzelmann P, Schindelin H, authors. The structural and functional basis of the p97/valosin-containing protein (VCP)-interacting motif (VIM): mutually exclusive binding of cofactors to the N-terminal domain of p97. J. Biol. Chem. 2011;286:38679–38690. [CrossRef] [PubMed]

Buchberger A, Schindelin H, Hanzelmann P, authors. Control of p97 function by cofactor binding. FEBS Lett. 2015;589:2578–2589. [CrossRef] [PubMed]

Falk S, et al., authors. Structure of the RBM7-ZCCHC8 core of the NEXT complex reveals connections to splicing factors. Nat. Commun. 2016;7:13573 [CrossRef] [PubMed]

Cote J, Richard S, authors. Tudor domains bind symmetrical dimethylated arginines. J. Biol. Chem. 2005;280:28476–28483. [CrossRef] [PubMed]

Friberg A, Oddone A, Klymenko T, Muller J, Sattler M, authors. Structure of an atypical tudor domain in the drosophila polycomblike protein. Protein Sci. 2010;19:1906–1916. [CrossRef] [PubMed]

Sattler M, Schleucher J, Griesinger C, authors. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. NMR Spectrosc. 1999;34:93–158. [CrossRef]

Farrow NA, et al., authors. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry. 1994;33:5984–6003. [CrossRef] [PubMed]

Grzesiek S, Bax A, authors. The importance of not saturating H₂O in protein NMR - application to sensitivity enhancement and NOE measurements. J. Am. Chem. Soc. 1993;115:12593–12594. [CrossRef]

Piotto M, Saudek V, Sklenář V, authors. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR. 1992;2:661 [CrossRef] [PubMed]

Vranken WF, et al., authors. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins. 2005;59:687–696. [CrossRef] [PubMed]

Winter G, author. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 2010;43:186–190. [CrossRef]

Winter G, et al., authors. DIALS: implementation and evaluation of a new integration package. Acta Crystallogr D. Struct. Biol. 2018;74:85–97. [CrossRef] [PubMed]

Potterton L, et al., authors. CCP4i2: the new graphical user interface to the CCP4 program suite. Acta Crystallogr. D. Struct. Biol. 2018;74:68–84. [CrossRef] [PubMed]

McCoy AJ, et al., authors. Phaser crystallographic software. J. Appl. Crystallogr. 2007;40:658–674. [CrossRef] [PubMed]

Emsley P, Lohkamp B, Scott WG, Cowtan K, authors. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 2010;66:486–501. [CrossRef] [PubMed]

Adams Paul D.; Afonine Pavel V.; Bunkóczi Gábor, Chen Vincent B.; Davis Ian W.; Echols Nathaniel, Headd Jeffrey J.; Hung Li-Wei, Kapral Gary J.; Grosse-Kunstleve Ralf W.; McCoy Airlie J.; Moriarty Nigel W.; Oeffner Robert, Read Randy J.; Richardson David C.; Richardson Jane S.; Terwilliger Thomas C.; Zwart Peter H., authors. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D Biological Crystallography. 2010;66(2):213–221. [CrossRef] [PubMed]

Painter J, Merritt EA, authors. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D. Biol. Crystallogr. 2006;62:439–450. [CrossRef] [PubMed]

Poser I, et al., authors. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods. 2008;5:409–415. [CrossRef] [PubMed]

Di Tommaso P, et al., authors. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 2011;39:W13–W17. [CrossRef] [PubMed]

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Fig. 1.

The N-terminal unstructured region of NVL interacts with the MTR4 KOW. a Schematic representation of the domain organization of NVL and MTR4. Domain boundaries are obtained from previous studies³¹,³⁷ and the crystal structure in the current report. The arrows indicate nomenclature for MTR4 constructs used in the text. b Protein co-precipitation assays. GST-tagged MTR4∆N, MTR4∆N∆arch, and MTR4 KOW were incubated with either Trx-NVL1–266 or Trx-NVL167–216 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes 1–8) and 30% of the eluates (lanes 9–16) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. c Microscale thermophoresis experiment with MTR4 KOW and Trx-NVL167–216-(GS)₃-eYFP. In all, 50 nM of Trx-NVL167–216-(GS)₃-eYFP was incubated with increasing concentrations of MTR4 KOW and thermophoresis was measured by tracking the fluorescence of the NVL-YFP fusion protein. The binding isotherm was calculated using MO software (Nanotemper technologies) and the dissociation constant (Kd) is given in the inset. The error bars represent the standard deviations of each data point calculated from three independent thermophoresis measurements

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Fig. 2.

Analysis of MTR4 KOW-NVL complex by NMR and site directed mutagenesis. a Overlay of ¹H-¹⁵N-HSQCs of either MTR4 KOW alone (black) or in complex with a six-fold excess of NVL2 (red). Selected residues experiencing large chemical shift perturbations (CSPs) are labeled. b Plot of CSPs per residue of the MTR4 KOW sequence. The red line marks the threshold of significant CSPs, which are mapped on the model of MTR4 KOW (PDB 6IEH) in panel c. Gaps indicate either prolines or residues that could not be assigned. c MTR4 KOW domain (PDB 6IEH) with labeled secondary structural elements as assigned by NMR and residues showing significant CSPs (displayed as red/green spheres). d Protein co-precipitations by pull down assays. GST-tagged MTR4∆N (WT or mutants) were incubated with Trx-NVL167–216 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes 1–4) and 30% of the eluates (lanes 5–8) were analyzed on 15% SDS-PAGE gels and visualized by staining with coomassie brilliant blue

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Fig. 3.

The vertebrate specific W-AIM in NVL is crucial for binding to MTR4 KOW. a Sequence alignment of vertebrate specific insertion regions of representative NVL sequences, Homo sapiens (Hs), Gallus gallus (Gg), Xenopus tropicalis (Xt), highlighting the W-AIM (GWFIDKTP) (red box), and the Nop53-like region (LFXϕD) (purple). The sequences were obtained from the UniProt database and aligned using the T-coffee server⁵⁹. b Protein co-precipitations by pull down assays. GST-tagged MTR4∆N was incubated with either wild type Trx-NVL167–216 or its indicated mutant before co-precipitation with glutathione sepharose beads. A total of 3% of the input (top) and 30% of the eluates (bottom) were analyzed on 15% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. c Overall structure of MTR4∆N₂70–1042 - NVL167–216 complex, with the DExH core of MTR4 colored in gray and arch colored in light blue. NVL is colored in orange. d Zoom-in view of the interactions between MTR4 KOW (light blue) domain and NVL (orange). Domains are colored as in Fig. 3a and viewed are 90° rotation along the horizontal and vertical axes with respect to the view in Fig. 3a. Residues discussed in the text are highlighted and labeled. e Cellular co-IP assay. FLAG-tagged wild type NVL construct, or its indicated mutant variant, were transiently expressed in cells stably expressing MTR4-LAP. After precipitation of MTR4-LAP, a total of 0.5% of the input (left) and 8.0% of the eluates (right) were analyzed on 4–12% SDS-PAGE gel followed by western blotting analysis. The primary antibody used is indicated below the panel

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Table 1.

Data collection and refinement statistics

| Mtr4ΔN70–1042 – NVL167–216
Data collection
 Space group | P6₁
 Cell dimensions |
   a, b, c (Å) | 184.37, 184.37, 90.53
   α, β, γ (°) | 90.0, 90.0, 120.0
 Resolution (Å) | 92.18–3.07
 Rsym or Rmerge | 15.7 (527.6)a
 I / σI | 12.6 (0.9)
 Completeness (%) | 99.9 (99.5)
 Redundancy | 16.5
 CC1/2 | 99.9 (62.7)
Refinement
 Resolution (Å) | 60.35–3.07 (3.22–3.07)
 No. reflections | 32,919
 Rwork/Rfree | 22.3 / 25.7
 No. atoms |
   Protein | 7074
   Ligand/ion | 58
   Water | 6
 B-factors |
   Protein | 123.57
   Ligand/ion | 138.03
   Water | 92.34
 R.m.s. deviations |
   Bond lengths (Å) | 0.003
   Bond angles (°) | 0.478

avalues in parentheses are for highest-resolution shell

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Fig. 4.

The N-terminus of ZCCHC8 interacts with the MTR4 KOW domain. a Schematic representation of the domain organization of ZCCHC8. Domain boundaries are obtained from previous studies³⁷,⁴³ and computational sequence analysis. b Protein co-precipitations by pull down assays. GST-tagged MTR4∆N, MTR4∆N∆arch, and MTR4 KOW were incubated with MBP-ZCCHC8 91–211 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes 1–4) and 30% of the eluates (lanes 5–8) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. c Microscale thermophoresis experiment with MTR4 KOW and MBP-ZCCHC8 91–211-(GS)₃-eYFP. In all, 1 μM MBP-ZCCHC8 91–211-(GS)₃-eYFP was incubated with increasing concentrations of MTR4 KOW and thermophoresis was measured by tracking the fluorescence of the ZCCHC8-YFP fusion protein. The binding curve was calculated using MO software (Nanotemper technologies) and the dissociation constant (Kd) is given in the inset. The error bars represent the standard deviations of each data point calculated from three independent thermophoresis measurements. d Competition experiment using GST pull down assay technology. A preformed GST-MTR4∆N:Trx-NVL167–216 complex was incubated with increasing concentrations of MBP-ZCCHC8(91–211) before precipitation with glutathione sepharose beads. A total of 3% of the input (lanes, 1–4) and 30% of the eluates (5–8) were analyzed on 15% SDS-PAGE gels and visualized by staining with coomassie brilliant blue

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Fig. 5.

Analysis of the ZCCHC8-MTR4 KOW complex by site directed mutagenesis. a Sequence alignment of the region between the predicted coiled coil domain and the zinc finger of ZCCHC8 from representative metazoan species, Homo sapiens (Hs), Gallus gallus (Gg), Xenopus laevis (Xl), highlighting NVL-like region (red), Nop53-like AIM (C-AIM) and ZCCHC8 specific I-AIM (purple boxes). The sequences were obtained from the UniProt database and aligned using the T-coffee server⁵⁹. b Protein co-precipitations by pull down assays testing ZCCHC8 91–211 mutants for MTR4∆N binding ability. GST-tagged MTR4∆N was incubated with either ZCCHC891–211 WT or mutants before co-precipitation with glutathione sepharose beads. A total of 3% of the input (top) and 30% of the eluates (bottom) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. c Protein co-precipitations by pull down assays testing MTR4∆N mutants for their ZCCHC8 91–211 binding ability. GST-tagged MTR4∆N (WT or mutant variants) were incubated with MBP-ZCCHC8 91–211 before co-precipitation with glutathione sepharose beads. A total of 3% of the input (lanes, 1–4) and 30% of the eluates (5–8) were analyzed on 12% SDS-PAGE gels and visualized by staining with coomassie brilliant blue. d Cellular co-IP assay. FLAG-tagged ZCCHC8 constructs (WT/IF mutant/CTD deletion) were transiently expressed in cells stably expressing MTR4-LAP. After precipitation of MTR4 taking advantage of the LAP tag, a total of 0.5% of the input (left) and 8.0% of the eluates (right) were analyzed on 4–12% SDS-PAGE gel followed by western blotting analysis. The primary antibody used is indicated below the panel

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